Temporal analysis of Arabidopsis genes activated by Eucalyptus grandis NAC transcription factors associated with xylem fibre and vessel development.

Secondary cell wall (SCW) deposition in Arabidopsis is regulated among others by NAC transcription factors, where SND1 chiefly initiates xylem fibre differentiation while VND6 controls metaxylem vessel SCW development, especially programmed cell death and wall patterning. The translational relevance of Arabidopsis SCW regulation theory and the utility of characterized transcription factors as modular synthetic biology tools for improving commercial fibre crops is unclear. We investigated inter-lineage gene activation dynamics for potential fibre and vessel differentiation regulators from the widely grown hardwood Eucalyptus grandis (Myrtales). EgrNAC26, a VND6 homolog, and EgrNAC61, an SND1 homolog, were transiently expressed in Arabidopsis mesophyll protoplasts in parallel to determine early and late (i.e. 7 and 14 hours post-transfection) gene targets. Surprisingly, across the time series EgrNAC26 activated only a subset of SCW-related transcription factors and biosynthetic genes activated by EgrNAC61, specializing instead in targeting vessel-specific wall pit and programmed cell death markers. Promoters of EgrNAC26 and EgrNAC61 both induced reporter gene expression in vessels of young Arabidopsis plants, with EgrNAC61 also conferring xylem- and cork cambium-preferential expression in Populus. Our results demonstrate partial conservation, with notable exceptions, of SND1 and VND6 homologs in Eucalyptus and a first report of cork cambium expression for EgrNAC61.

Enhanced beta cell proliferation in mice overexpressing a constitutively active form of Akt and one allele of p21Cip.

AIMS/HYPOTHESIS: The ability of pancreatic beta cells to proliferate is critical both for normal tissue maintenance and in conditions where there is an increased demand for insulin. Protein kinase B(Akt) plays a major role in promoting proliferation in many cell types, including the insulin-producing beta cells. We have previously reported that mice overexpressing a constitutively active form of Akt(caAkt (Tg)) show enhanced beta cell proliferation that is associated with increased protein levels of cyclin D1, cyclin D2 and cyclin-dependent kinase inhibitor 1A (p21(Cip)). In the present study, we sought to assess the mechanisms responsible for augmented p21(Cip) levels in caAkt(Tg) mice and test the role of p21(Cip) in the proliferative responses induced by activation of Akt signalling. METHODS: To gain a greater understanding of the relationship between Akt and p21(Cip), we evaluated the mechanisms involved in the modulation of p2(Cip) by Akt and the in vivo role of reduced p21(Cip) in proliferative responses induced by Akt. RESULTS: Our experiments showed that Akt signalling regulates p21(Cip) transcription and protein stability. caAkt(Tg) /p21(Cip+/-) mice exhibited fasting and fed hypoglycaemia as well as hyperinsulinaemia when compared with caAkt(Tg) mice. Glucose tolerance tests revealed improved glucose tolerance in caAkt(Tg)/p21(Cip+/-) mice compared with caAkt (Tg). These changes resulted from increased proliferation, survival and beta cell mass in caAkt(Tg)/p21(Cip+/-) compared with caAkt(Tg) mice. CONCLUSIONS/INTERPRETATION: Our data indicate that increased p21(Cip) levels in caAkt(Tg) mice act as a compensatory brake, protecting beta cells from unrestrained proliferation. These studies imply that p21(Cip) could play important roles in the adaptive responses of beta cells to proliferate in conditions such as in insulin resistance.

Beta-cell failure as a complication of diabetes.

Type 2 diabetes mellitus is a complex disease characterized by beta-cell failure in the setting of insulin resistance. In early stages of the disease, pancreatic beta-cells adapt to insulin resistance by increasing mass and function. As nutrient excess persists, hyperglycemia and elevated free fatty acids negatively impact beta-cell function. This happens by numerous mechanisms, including the generation of reactive oxygen species, alterations in metabolic pathways, increases in intracellular calcium and the activation of endoplasmic reticulum stress. These processes adversely affect beta-cells by impairing insulin secretion, decreasing insulin gene expression and ultimately causing apoptosis. In this review, we will first discuss the regulation of beta-cell mass during normal conditions. Then, we will discuss the mechanisms of beta-cell failure, including glucotoxicity, lipotoxicity and endoplasmic reticulum stress. Further research into mechanisms will reveal the key modulators of beta-cell failure and thus identify possible novel therapeutic targets. Type 2 diabetes mellitus is a multifactorial disease that has greatly risen in prevalence in part due to the obesity and inactivity that characterize the modern Western lifestyle. Pancreatic beta-cells possess the potential to greatly expand their function and mass in both physiologic and pathologic states of nutrient excess and increased insulin demand. beta-cell response to nutrient excess occurs by several mechanisms, including hypertrophy and proliferation of existing beta-cells, increased insulin production and secretion, and formation of new beta-cells from progenitor cells [1, 2]. Failure of pancreatic beta-cells to adequately expand in settings of increased insulin demand results in hyperglycemia and diabetes. In this review, we will first discuss the factors involved in beta-cell growth and then discuss the mechanisms by which beta-cell expansion fails and leads to beta-cell failure and diabetes (Fig. 1).

Mice with beta cell overexpression of glycogen synthase kinase-3beta have reduced beta cell mass and proliferation.

AIMS/HYPOTHESIS: Glycogen synthase kinase-3 (GSK3) has been implicated in the pathophysiology of several prevalent diseases, including diabetes. However, despite recent progress in our understanding of the role of GSK3 in the regulation of glucose metabolism in peripheral tissues, the involvement of GSK3 in islet beta cell growth and function in vivo is unknown. We therefore sought to determine whether over-activation of GSK3beta would lead to alterations in islet beta cell mass and/or function. METHODS: Transgenic mice overexpressing a constitutively active form of human GSK3beta (S9A) under the control of the rat insulin promoter (RIP-GSK3betaCA) were created. Studies using mouse insulinoma cells (MIN6) were conducted to investigate the regulation of GSK3beta activity and its impact on pancreas/duodenum homeobox protein-1 (PDX-1) levels. RESULTS: We demonstrated that phosphorylation of GSK3beta was decreased, indicating increased GSK3beta activity in two animal models of diabetes, Lepr(-/- ) mice and Ins2 (Akita/+) mice. In MIN6 cells, the activity of GSK3beta was regulated by glucose, in a fashion largely dependent on phosphatidylinositol 3-kinase. RIP-GSK3betaCA transgenic mice showed impaired glucose tolerance after 5 months of age. Histological studies revealed that transgenic mice had decreased beta cell mass and decreased beta cell proliferation, with a 50% decrease (p<0.05) in the level of PDX-1. CONCLUSIONS/INTERPRETATION: We showed direct evidence that GSK3beta activity is associated with beta cell failure in diabetic mouse models and that its overactivation resulted in decreased pancreatic beta cell proliferation and mass. GSK3 modulates PDX-1 stability in both cultured insulinoma cells and islets in vivo. These results may ultimately facilitate the development of potential therapeutic interventions targeting type 2 diabetes and/or islet transplantation.

Regulation of beta-cell mass and function by the Akt/protein kinase B signalling pathway.

The insulin receptor substrate-2/phosphoinositide 3-kinase (PI3K) pathway plays a critical role in the regulation of beta-cell mass and function, demonstrated both in vitro and in vivo. The serine threonine kinase Akt is one of the promising downstream molecules of this pathway that has been identified as a potential target to regulate function and induce proliferation and survival of beta cells. Here we summarize some of the molecular mechanisms, downstream signalling pathways and critical components involved in the regulation of beta-cell mass and function by Akt.

Mice conditionally lacking the Wolfram gene in pancreatic islet beta cells exhibit diabetes as a result of enhanced endoplasmic reticulum stress and apoptosis.

AIMS/HYPOTHESIS: Wolfram syndrome is an autosomal recessive disorder characterised by childhood diabetes mellitus, optic atrophy and severe neurodegeneration, resulting in premature death. The aim of this study was to investigate the mechanisms responsible for the phenotype of carbohydrate intolerance and loss of pancreatic beta cells in this disorder. MATERIALS AND METHODS: To study the role of the Wolfram gene (Wfs1) in beta cells, we developed a mouse model with conditional deletion of Wfs1 in beta cells by crossing floxed Wfs1 exon 8 animals with mice expressing Cre recombinase under the control of a rat insulin promoter (RIP2-Cre). Complementary experiments using RNA interference of Wfs1 expression were performed in mouse insulinoma (MIN6) cell lines (WfsKD). RESULTS: Male knockout mice (betaWfs(-/-)) began developing variable and progressive glucose intolerance and concomitant insulin deficiency, compared with littermate controls, by 12 weeks of age. Analysis of islets from betaWfs(-/-) mice revealed a reduction in beta cell mass, enhanced apoptosis, elevation of a marker of endoplasmic reticulum stress (immunoglobulin heavy chain-binding protein [BiP]), and dilated endoplasmic reticulum with decreased secretory granules by electron microscopy. WfsKD cell lines had significantly increased apoptosis and elevated expression of the genes encoding BiP and C/EBP-homologous protein (CHOP), two markers of endoplasmic reticulum stress. CONCLUSIONS/INTERPRETATION: These results indicate that (1) lack of expression of Wfs1 in beta cells was sufficient to result in the diabetes mellitus phenotype; (2) beta cell death occurred by an accelerated process of apoptosis; and (3) lack of Wfs1 was associated with dilated endoplasmic reticulum and increased markers of endoplasmic reticulum stress, which appears to be a significant contributor to the reduction in beta cell survival.

An expression profile of human pancreatic islet mRNAs by Serial Analysis of Gene Expression (SAGE).

AIMS/HYPOTHESIS: The Human Genome Project seeks to identify all genes with the ultimate goal of evaluation of relative expression levels in physiology and in disease states. The purpose of the current study was the identification of the most abundant transcripts in human pancreatic islets and their relative expression levels using Serial Analysis of Gene Expression. METHODS: By cutting cDNAs into small uniform fragments (tags) and concatemerizing them into larger clones, the identity and relative abundance of genes can be estimated for a cDNA library. Approximately 49,000 SAGE tags were obtained from three human libraries: (i) ficoll gradient-purified islets (ii) islets further individually isolated by hand-picking, and (iii) pancreatic exocrine tissue. RESULTS: The relative abundance of each of the genes identified was approximated by the frequency of the tags. Gene ontology functions showed that all three libraries contained transcripts mostly encoding secreted factors. Comparison of the two islet libraries showed various degrees of contamination from the surrounding exocrine tissue (11 vs 25%). After removal of exocrine transcripts, the relative abundance of 2180 islet transcripts was determined. In addition to the most common genes (e.g. insulin, transthyretin, glucagon), a number of other abundant genes with ill-defined functions such as proSAAS or secretagogin, were also observed. CONCLUSION/INTERPRETATION: This information could serve as a resource for gene discovery, for comparison of transcript abundance between tissues, and for monitoring gene expression in the study of beta-cell dysfunction of diabetes. Since the chromosomal location of the identified genes is known, this SAGE expression data can be used in setting priorities for candidate genes that map to linkage peaks in families affected with diabetes.

Islet beta cell expression of constitutively active Akt1/PKB alpha induces striking hypertrophy, hyperplasia, and hyperinsulinemia.

The phosphoinositide 3-kinase-Akt/PKB pathway mediates the mitogenic effects various nutrients and growth factors in cultured cells. To study its effects in vivo in pancreatic islet beta cells, we created transgenic mice that expressed a constitutively active Akt1/PKB alpha linked to an Insulin gene promoter. Transgenic mice exhibited a grossly visible increase in islet mass, largely due to proliferation of insulin-containing beta cells. Morphometric analysis verified a six-fold increase in beta cell mass/pancreas, a two-fold increase in 5-bromo-2'-deoxyuridine incorporation, a four-fold increase in the number of beta cells per pancreas area, and a two-fold increase in cell size in transgenic compared with wild-type mice at 5 weeks. At least part of the increase in beta cell number may be accounted for by neogenesis, defined by criteria that include beta cells proliferating from ductular epithelium, and by a six-fold increase in the number of single and doublet beta cells scattered throughout the exocrine pancreas of the transgenic mice. Glucose tolerance was improved, and fasting as well as fed insulin was greater compared with wild-type mice. Glucose-stimulated insulin secretion was maintained in transgenic mice, which were resistant to streptozotocin-induced diabetes. We conclude that activation of the Akt1/PKB alpha pathway affects islet beta cell mass by alteration of size and number.

Activation of Elk-1, an Ets transcription factor, by glucose and EGF treatment of insulinoma cells.

Elk-1, a member of the ternary complex factor family of Ets domain proteins that bind serum response elements, is activated by phosphorylation in a cell-specific manner in response to growth factors and other agents. The purpose of the current study was to determine whether Elk-1 activation contributes to glucose-/depolarization-induced Ca(2+)-dependent induction of immediate early response genes in pancreatic islet beta-cells. The results of experiments in insulinoma (MIN6) cells demonstrated that Elk-1-binding sites (Ets elements) in the Egr-1 gene promoter contribute to transcriptional activation of the gene. Treatment with either epidermal growth factor (EGF), a known inducer of beta-cell hyperplasia, glucose, or KCl-induced depolarization resulted in Ser(383) phosphorylation and transcriptional activation of Elk-1 (4 +/- 0.3-, P = 0.003, 2.3 +/- 0.19-, P = 0.002, and 2.2 +/- 0.1- fold, P = 0.001 respectively). The depolarization response was inhibited by the Ca(2+) channel blocker verapamil and by the MEK inhibitor PD98059 (53 +/- 6 and 55 +/- 0.5%, respectively). EGF-induced activation of Elk-1 was also inhibited by PD98059 (60 +/- 5%). A dominant negative Ras produced partial inhibition (42%) of the depolarization-induced Elk-1 transcriptional activation. Transfection with a constitutively active Ca(2+)/calmodulin kinase IV plasmid also resulted in Elk-1 transcriptional activation. Experiments with p38, phosphatidylinositol 3-kinase, and protein kinase A inhibitors indicated that these pathways are not involved. We conclude that Elk-1 activation contributes to glucose-/depolarization-induced Ca(2+)-dependent induction of immediate early growth response genes in pancreatic islet beta-cells. Furthermore, the results demonstrated a convergence of nutrient- and growth factor-mediated signaling pathways on Elk-1 activation through induction of Ras/mitogen-activated protein kinase ERK-1 and -2. The role of these pathways in the glucose-induced proliferation of islet beta-cells can now be assessed.

Glucose and other insulin secretagogues induce, rather than inhibit, expression of Id-1 and Id-3 in pancreatic islet beta cells.

AIMS/HYPOTHESIS: Basic helix loop helix transcription factors regulate insulin gene transcription. Therefore, molecules that regulate their function should affect insulin production and secretion. As Id proteins inhibit basic helix loop helix function, it is important to determine whether they are expressed in beta cells and if insulin secretagogues regulate their expression. METHODS: Human islets or insulinoma cells were cultured in different glucose concentrations or treated with secretagogues. Insulin secretion was measured using RIA. The Id mRNA and protein concentrations were measured using northern blots, RT-PCR, and western blots. Transfections of promoter-reporter constructs were used to estimate Id-1 gene transcription. RESULTS: The Id-1 mRNA concentrations were twofold higher in islets cultured overnight in 10 mmol/l than in 2.5 mmol/l glucose. Addition of high glucose to islets previously cultured in low glucose, increased Id-1 mRNA concentrations within 30 min. Analyses using insulinoma cells revealed that Id-1 and Id-3 mRNA concentrations peaked 30 min after glucose was added, returned to near basal concentrations by 2 h and then progressively increased for 24 h. The Id-1 protein concentrations changed in a similar pattern. Insulin secretagogues that act through different signaling pathways also induced Id expression. The Id response required glucose metabolism, calcium, and RNA synthesis but not protein synthesis. Glucose-responsive elements are confined to the 5'-region of the Id-1 gene. CONCLUSION/INTERPRETATION: The concomitant induction of Id-1 and Id-3 expression, insulin gene transcription, and insulin secretion suggests that physiological concentrations of Ids do not inhibit insulin gene transcription and Ids could play unexpected and novel roles in promoting beta-cell function.

Activation of serum response factor in the depolarization induction of Egr-1 transcription in pancreatic islet beta-cells.

The results of the current studies define the major elements whereby glucose metabolism in islet beta-cells leads to transcriptional activation of an early response gene in insulinoma cell lines and in rat islets. Glucose stimulation (2-20 mm) resulted in a 4-fold increase in Egr-1 mRNA at 30 min, as did the depolarizing agents KCl and tolbutamide. This response was inhibited by diazoxide and EGTA, indicating that beta-cell depolarization and Ca(2+) influx, respectively, are essential. Pharmacological inhibition of the Egr-1 induction by H89 (48%) and calmidazolium (35%), but not by mitogen-activated protein kinase/extracellular signal-regulated kinase kinase 1 and 2 or phosphatidylinositol 3-kinase inhibitors, implied that protein kinase A and Ca(2+)/calmodulin pathways are involved. Deletion mapping of the Egr-1 promoter revealed that the proximal -198 base pairs containing two serum response elements (SREs) and one cAMP-response element retained the depolarization response. Depolarization resulted in phosphorylation of cAMP-response element-binding protein, yet partial inhibition by a dominant negative cAMP-response element-binding protein, along with a robust response of a cAMP-response element-mutated Egr-1 promoter suggested the presence of a second Ca(2+)-responsive element. Depolarization activation of 5XSRE-LUC and serum response factor (SRF)-GAL4 constructs, along with activation of SRF-GAL4 by co-transfection with constitutively active calmodulin kinase IV and protein kinase A, and binding of Ser(103)-phosphorylated SRF in nuclear extracts, indicated that the SRE.SRF complexes contribute to the Ca(2+)-mediated transcriptional regulation of Egr-1. The results of the current experiments demonstrate for the first time SRE-dependent transcription and the role of SRF, a transcription factor known to be a major component of growth responses, in glucose-mediated transcriptional regulation in insulinoma cells.

A gene encoding a transmembrane protein is mutated in patients with diabetes mellitus and optic atrophy (Wolfram syndrome).

Wolfram syndrome (WFS; OMIM 222300) is an autosomal recessive neurodegenerative disorder defined by young-onset non-immune insulin-dependent diabetes mellitus and progressive optic atrophy. Linkage to markers on chromosome 4p was confirmed in five families. On the basis of meiotic recombinants and disease-associated haplotypes, the WFS gene was localized to a BAC/P1 contig of less than 250 kb. Mutations in a novel gene (WFS1) encoding a putative transmembrane protein were found in all affected individuals in six WFS families, and these mutations were associated with the disease phenotype. WFS1 appears to function in survival of islet beta-cells and neurons.

The inhibition of the reverse transcriptase of HIV-1 by the natural sulfoglycolipids from cyanobacteria: contribution of different moieties to their high potency.

The potent in vitro inhibition of the enzymatic activity of the human immunodeficiency virus-1 (HIV-1) reverse transcriptase (RT) by the lipophilic extracts of cyanobacteria8 was primarily attributed to the sulfoquinovosylpranosyl lipids, compounds 1-4. These sulfolipids inhibit efficiently and selectively only the DNA polymerase activity of HIV-1 RT (and not the ribonuclease H function) with 50% inhibitory concentration value (IC50) as low as 24 nM exhibited by compound 1. The novel natural compound 4, in which two hydroxy groups on the sugar moiety are substituted by palmitoyl residues, exhibits a significant decrease in the maximal inhibition capacity. It is possible, therefore, that the contribution of acylated groups to the molecule at these positions interferes with inhibition, possibly, by steric hindrance. Both the sulfonic acid moiety and the fatty acid ester side chain have a substantial effect in potentiating the extent of inhibition. For one, the inhibitory effects of all the natural glycolipids tested (5-8) are markedly reduced, and the hydrolysis of the fatty acid side chain, as in derivative 9, has substantially abolished the inhibition of HIV RT.

Somatosensory evoked potentials of the posterior tibial nerve in hemiparetic patients: relation to stance balance and walking ability.

OBJECTIVES: To examine the association between stance ability and walking performance of poststroke hemiplegic patients and their posterior tibial nerve somatosensory evoked potentials (SEPs). DESIGN AND SETTING: Fifteen patients, residents of a geriatric rehabilitation hospital, were evaluated twice, with a 2-week interval between sessions. In each session, clinical tests of stance balance and walking ability were performed, and bilateral SEPs to stimulation of the posterior tibial nerve were recorded. Eight healthy, age-matched control subjects underwent the same tests in a single session, but SEPs were recorded unilaterally. Correlation analysis and analysis of variance (ANOVA) were used for studying the prognostic value of the initial posterior tibial nerve SEP measurements as well as the within- and between-sessions relationships between the clinical-functional tests and the SEP data. RESULTS: No significant correlations between the initial SEP values and functional improvement were established. Within each session, positive significant correlations existed between decreased latencies of several of the medium-latency SEP waves and the performance of stance and gait tasks. However, the between-sessions improvement in stance balance was not correlated with a decrease in latency of the SEP peaks or with an increase in their amplitudes. As to walking ability, in those patients whose gait significantly improved, a significant shortening of P37 and P54 latencies took place. CONCLUSIONS: The association between the initial and/or the 2-week changes in SEP of the posterior tibial nerve and improvement in stance and walking abilities is equivocal. In addition, the applicability of SEP measurements is limited by patients' physical status and cooperation. The clinical significance of posterior tibial nerve SEP testing in poststroke hemiparetic patients is therefore debatable.

New acylated sulfoglycolipids and digalactolipids and related known glycolipids from cyanobacteria with a potential to inhibit the reverse transcriptase of HIV-1.

Five novel diacylated sulfoglycolipids (1-5) were isolated from the cyanobacterium Scytonema sp. (TAU strain SL-30-1-4) and four novel acylated diglycolipids (6-9) were isolated from the cyanobacterium Oscillatoria raoi (TAU strain IL-76-1-2). These two groups of glycolipids and related known glycolipids isolated from these two and three other strains of cyanobacteria, Phormidium tenue (TAU strain IL-144-1), O. trichoides (TAU strain IL-104-3-2), and O. limnetica (TAU strain NG-4-1-2), were found to inhibit HIV-1 RT enzymatic activity to different extents. The structure elucidation of the various compounds is based on the selective hydrolysis of the glycerol ester moieties, GCMS analysis of the methyl ester derivatives of the liberated fatty acids, homo- and heteronuclear-2D-NMR techniques, and MS. The use of negative-ion FABMS for analyzing the combination and distribution of the fatty acids in glycolipids is demonstrated.

Prevalence of viral antibodies in gingival crevicular fluid.

The prevalence of antibodies to CMV, Mumps and Coxsackie virus strains 1, 3 and 4 was studied in 39 samples of gingival crevicular fluids (GCF) obtained from clinical healthy patients and compared to the corresponding antibodies present in the serum of each individual. In spite of the high prevalence of humoral antibodies to CMV (75%), only 24% of the gingival crevicular fluid samples exhibited IgG or IgA antibodies to this virus. The differences in the prevalence of antibodies against Mumps virus in the sera and GCF were even greater: whereas 87% of the patients exhibited serum antibodies, not even a single gingival fluid sample was found to be positive. Antibodies to Coxsackie B strains 1, 3 and 4 were found in 72%, 63% and 52% of the sera and in 25%, 19% and 33% of the gingival fluid samples (IgG only). The presence of the antibodies and their profile in GCF and serum is different. The mechanism of possible permeation is not clear but it seems that viral antibodies in this milieu are not derived from the serum solely by passive transudation, and that the antibodies are produced locally at least in some of the GCF specimens.

The effect of vagolytic drugs on airway obstruction in patients with bronchial asthma.

Atropine sulphate and ipratropium bromide (Atrovent, Sch-1000 were found to be potent bronchodilators free from significant side effects. Their action was similar in magnitude to the one produced by salbutamol. Both drugs were particularly effective at the level of the large airways.